Label-free and quantitative analysis of C-reactive protein in human sera by tagged-internal standard assay on antibody arrays.
We have developed a new, high-throughput, competition-based tagged-internal standard (TIS) assay to measure the levels of blood proteins in human serum. In this assay, target proteins in the sample serum compete with tagged-internal standard proteins for binding to an antibody array. Antibody arrays are fabricated by immobilizing a target protein-specific antibody on the carboxylate-modified latex bead surface of well-type arrays. A solution of Alexa 546-conjugated target protein is added to a sample of human serum and applied to the well-type antibody array. The array is then analyzed with a fluorescence scanner and the level of unlabeled target protein in the human sera is inferred from the amount of tagged protein bound to the array. We successfully applied this assay to measure the level of C-reactive protein (CRP) in 92 unlabeled human sera. The TIS assay was found to be specific and reproducible for the quantitative analysis of CRP. The antibody array data from the TIS assay correlate well with clinical laboratory data obtained using the commercialized latex-enhanced turbidimetry immunoassay (n=3, r=0.967, CV=0.32%). Thus, the antibody array-based TIS assay system is high-throughput, quantitative, and label-free and may be useful in the rapid serodiagnosis of human disease.
Jung
Jae-Wan
JW
Department of Molecular and Cellular Biochemistry and Vascular System Research Center, Chuncheon, Kangwon-Do 200-701, South Korea.
Jung
Se-Hui
SH
Yoo
Je-Ok
JO
Suh
In-Bum
IB
Kim
Young-Myeong
YM
Ha
Kwon-Soo
KS
D000906:Antibodies / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D001774:Blood Chemical Analysis / Q000295:instrumentation*; D002097:C-Reactive Protein / Q000032:analysis* / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D040081:Protein Array Analysis / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D000906:Antibodies; D002097:C-Reactive Protein